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Lab Partners: Mariah Blecher, Keith Frey, Tyser Lyfoung, Sofia Rahmanzai
Introduction:
The goal of the overall experiment is to figure out the genome sequence of the virus
found in the water sample. The specific goal in the part of the lab is to figure out different
characterizations of the virus such as virus type, size, concentration and other factors about the
virus in the water sample. Figuring out these characterizations will help further our knowledge
First we need to isolate our viral genome. Nucleic acid extraction is done to get the
isolated viral genome. Extraction is used to digest contaminating cellular nucleic acid before the
virus genome is extracted from the viral capsid. Digestion of these capsid proteins are done using
phenol and phenol chloroform followed by ethanol precipitation to recover viral nucleic acids
and genome. After this process, characterization of the genome can be taken with the Nanodrop
2000. Only a small drop is needed to use the Nanodrop 2000. It takes the concentration of the
nucleic acid in the sample by using UV light. The software will do all the calculations to
Restriction of the viral DNA is then done by performing an agarose gel electrophoresis.
Restriction allows certain DNA sequences to be recognized and then make scissions in the
DNA. In this experiment this screening is done on unknown viral genome. These are done in
diagnostic restriction digest. This will be done by agarose gel electrophoresis. This method is
through the gel. Smaller molecules will also move faster than the larger ones, so these bands can
actually be read. This is done in many different lab techniques to characterize size of DNA and
proteins. The gel is prepared and set. A comb is used to create wells to where the sample can be
put into. Positive and negative electrodes are attached, one on each end to create the electrical
Another method to characterize viruses can be done by using electron microscopy. This is
a very amazing technique and was done at UNMC. The sample was negatively stained and then
scanned through to find viruses. This gives us insight on how the virus looks and what shape and
type the virus may be. This is done by staining the virus and shining a beam of light through it to
get clear images under the microscope. Then measurements can be taken and pictures taken as
well.
Methods:
Remove two aliquots of 0.5 ml of viral stock into separate eppendoft tubes. Then, 5 l of
DNase I and 5 l of RNase A was added. This was incubated for 30 minutes at room
temperature. Then after incubation, 5 l of proteinase K and 25 l of SDS was added to each
Next is phenol extraction and ethanol precipitation after incubation of an hour. This is all
done in the hood due to how dangerous phenol is. Gloves and eye protection were to be worn as
well. To this mixture, 500 l of phenol was added, vortexed 30 seconds and then separated into
phases by centrifugation for 2 minutes. After 2 minutes, remove the upper aqueous phase into a
new microfuge tube. Then add 500 l of phenol-chloroform, vortex for 30 seconds and then
centrifugation for 2 minutes. Again, transfer the upper aqueous phase into a new tube. This time,
add 500 l of chloroform, vortex for 30 seconds and centrifuge for 2 minutes. Again transfer the
95% ethanol to this tube. Mix by inverting and observe the solution. Store overnight at -20C. To
recover the nucleic acid, centrifuge in a microfuge for 10 minutes at 4C. Discard the supernatant
and was the pellet with 600 l of cold 70% ethanol without mixing. Centrifuge for 5 minutes in a
microfuge. Discard the supernatant, dry the pellet for 2 minutes and resuspend the pellet in 50 l
*Note: this was done twice; once on 2/22/17 and 3/8/17. Readings of the Nano 2000 were
taken 3/8/17
Gel Electrophoresis
TAE Buffer
- To make 10X TAE buffer with 5 ug/ml EthBr, dilute 10 ml of 50X to 50 ml then 25 l
mg/ml EthBr stock was added. This was the gel running buffer.
flask. The agarose was completely melted in the microwave and boiled to dissolve the agarose.
More water was added to replace the water that was boiled away. Then 5 ml of 10X TAE buffer
containing 5ug/ml Ethidium Bromide was added. The solution was cooled to less than 60C then
poured into the gel tray. A comb was placed into the gel to form the wells and the gel then
solidified.
To load the gel, a total of 10 l was loaded. Using the concentration determined from the
Nano 2000, we wanted 1 g of the nucleic acid so we determined to use 3.5 l of our nucleic
acid, 2 l of loading dye and 4.5 l of water. The specs on the machine was the following: time-
59, volts- 69 and mA- 49. The positive end of the electrode was orientated away from the wells.
Negative Staining:
This was done at UNMC in their lab. A 30 l drop was moved onto parafilm.
With a Formvar and carbon coated copper grid is floated with a coated surface on the drop of
suspension for 1 minute then the excess was wiped away and air dried for 30 seconds. Then 30
l of negative staining solution was put onto the film. The grid with sample absorbed to the
surface is floated on the drop of negative stain for 2 minutes and again excess was wiped off and
hair tired for 60 minutes. Then the grid is examined by the transmission electron microscope.
Results:
Strands of visible DNA wasnt noticeable until a week later when it was checked. There
Readings for the Nanodrop were recorded March 8th, 2017 using the nucleic acid
extraction. In figure 1 below, there is a slope at 260 nm which shows that there is nucleic acids in
our solution. The concentration was read at 281.3 ng/l. The ratio of nucleic acids is 1.77.
Gel Electrophoresis
The agarose gel electrophoresis was processed on March 8th, 2017. Looking at lane 4,
there is a very faint line towards the top. Perhaps the nucleic acid didnt pick up enough stain to
1 2 3 4 5 6 7 8
+ end
Figure 2: Agarose gel. Lane 4 is group 3 and there is a very faint line towards the top.
Electron Microscopy
According to the pictures and measurements, the head length is 78.26-91.48 nm long, the
width of the head is 68.05-78.18 nm wide, tail length is 123.9 nm long and tail width is 20.76 nm
wide. The average head length is about 75 nm and 78 nm wide. Photographs are at 110,000 times
Picture 3: appears to be infecting a particle. Picture 4: measurements of head and also of the
head.
Discussion:
According to all our observations, Small Private B is a bacteriophage. After looking at the
microscopy, it appears to a bacteriophage with its distinctive head and tail shape.
Later we will do a one-step growth curve for our virus. Still want to keep trying SDS-PAGE. We